Inhibitory effect of synthetic and natural colorants on carcinogenesis

ABSTRACT

A method of reducing the incidence of pulmonary tumor formation in animals exposed to a tumor-promoting chemical or a tumor initiating by providing a group of animals which have been exposed to a tumor-promoting chemical or a tumor-initiating chemical with drinking water containing betanins. Preferably, the betanins are obtained from a beetroot extract.

This application is a divisional application of U.S. application Ser.No. 08/845,166, filed Apr. 21, 1997, which in turn claims the benefit ofU.S. Provisional application Ser/ No. 60/022,638, filed Jul. 24, 1996.

FIELD OF THE INVENTION

The field of the invention relates to use of food colorants in thetreatment or prevention of cancer. More particularly, the inversionrelates to use of safe, nontoxic food colorants to inhibitcarcinogenesis. The food colorants may be natural pigments extractedfrom a plant or plants, or they may be synthetic compounds approved bythe FDA for use as food colorants or food dyes.

BACKGROUND OF THE INVENTION

A wide variety of colorants have been approved by the FDA for use infoods, pharmaceuticals, and cosmetic preparations. Extensive toxicitystudies have been performed on each of these compounds, and they havebeen found to be nontoxic. However, relatively little research has beenperformed to determine whether use of these colorants in foods, andcosmetics is actually beneficial, rather than simply not harmful.

We have been conducting studies designed to help fill this gap. Ourresearch is directed towards use of natural and synthetic food colorantsin the inhibition of carcinogenesis. In past work, we have describedinhibition of tumor formation by natural colorants and and extracts offruits, vegetables, and other plant parts (Kapadia et al., Proc. IntCongress on Nat. Prod. Research, Abstract No. 225; Halifax, Canada;1994). Extracts of turmeric, paprika, and annatto seeds have been foundto be of great interest, particularly in the prevention of cancer.Paprika extracts, obtained from Capsicum anmum L. (Fam: solanacaea), areamong the oldest and most important carotenoid extracts. The maincarotenoids present in paprika are capsanthin (I) and capsorubin (II),largely as their acyl esters. Other minor components are present.

Annatto seeds are the seeds of the tropical bush Bixa orellana L.(Family: Bixaceae). The major pigment is the carotenoid cis-bixin (III),the monomehyl ester of diapocarotenoic acid (IV, also known ascis-norbixin). Trans-bixin and cis-norbixin are also present as minorcostituents. Trans-bixin and cis-bixin are both sparingl water-soluble,and may be converted to a water-soluble salt of the correspondingnorbixin upon alkali hydrolysis.

Turmeric, also called curcuma, is a fluorescent yellow-colored extractfrom the rhizomes of seven species of the curcuma plant. Curcuma longais the usual commercial source. The pigments curcumin (V),dernethoxycurcumin (VI), and bisdemethoxycurcumin (VII) occur in everyspecies, together with minor ingredients that contribute to the flavor.

Other natural colorants that have been found to be of interest includethe betanins and the anthocyanins. Betanins may be readily extractedfrom beet roots, and anthocyanis may be extracted from grapes, red onionskin, cranberries, and other plant-derived materials.

The studies reported herein include in vitro studies of inhibition ofEpstein-Barr virus early antigen (EBV-EA) induction studies by synthdiccolorants and by natural plant extracts and colorants. The Epstein-Barrvirus (EBV)is a member of the herpes virus family, and to beenimplicated in the pathogenesis of Burkitt's lymphoma, nasopharyngealcarcinoma, and B lymphocyte neoplasms. Infection of a human by EBV iscommonly followed by a latency period, when the virus is presentintracellularly in an unexpressed state. T cell suppressor mechanisms ofthe immune response to EBV are effective in the inhibition of acute EBVinfection. If these mechanisms fail, Burkitt's lymphoma, nasopharyngealcarcinoma, and B cell lymphomas may emerge. This is a serious problemduring the postoperative recuperation period following organtransplants, as immunosuppresive therapy is often used to preventrejection. It is therefore important to find new cancer chemopreventiveagents which prevent formation of tumors linked to the Epstein-Barrvirus. In vitro inhibition of EBV-EA induction in Epstein-Barr virusgenome-carrying cells by a potential cancer chemopreventive compoundserves as an excellent sign that the compounds may inhibit tumorformation in a living organism. In fact, inhibitory activity towardEBV-EA induction is often taken as a measure of anti-tumor promotingactivity.

In vivo studies of inhibition of skin tumor formation by synthetic andnatural colorants were also performed, using a two-stage mouse skincarcinogenesis test. A diagram illustrating such a test is provided inFIG. 1. Studies on rats and mice provide insight into the cancerinduction process in humans and animals. Two mice (or groups of mice)are each treated with a compound known to initiate tumor formation(stage 1). In FIG. 1, 7,12-methylbenz[α]anthracene (DMBA) is used as thetumor initiator; other compounds may be used. The mice are thensubjected to a condition or compound on a continued basis that promotestumor formation (stage 2). Ultraviolet B radiation serves as aneffective tumor promoter, as shown in FIG. 1. Topical application of12-O-tetradecanoylphorbol-13-acetate (TPA) also serves as a promoter ofskin tumors. One animal, the test animal, is provided an inhibitor oftumor promotion. The inhibitor may be provided in drinking water ortopically applied to the skin. The other animal, the control animal, isprovided with no inhibitor. At the end of a defined period, the numberof tumors on the test animal is compared to the number of tumors on thecontrol animal. Similar tests were performed for inhibition of pulmonarytumor formation, using injected 4-nitroquinoline 1-oxine as a tumorinitiator and glycerol, provided in drinking water as a tumor promoter.

In EMF Health Report, vol. 1, No. 2 (1993), cancer initiators aredefined as compounds that cause genetic damage, Only a single exposureis required to start the cancer-forming progress. Compounds whichinitiate tumor formation include nitrosamines, alkylating agents,aromatic amines, polycyclic aromatic hydrocarbons,dimethylbenz[α]anthracene, vinyl chloride, asbestos,N-methyl-N-nitrosourea, and azaserine, as disclosed in Advances inCancer Research, vol. 50, pp. 26-30.

In EWF Health Report, vol. 1, No, 2 (1993), cancer promoters are definedas compounds that cause irritation, inflammation, and cell growth whenapplied to an animal alone, and which increase the rate of tumor growthwhen applied after exposure to an initiator. Compounds which initiatetumor formation include active phorbol esters, such astetradecanoylphorbol acetate and phorbol dibenzoate; teleocidin andrelated compounds; aplysiatoxin and related compounds; mezerein;tetradecanoyl ingenol; iodoacetic acid; benzoyl peroxide; palytoxin; andanthralin. These promoters are disclosed in Biomedicine &Pharmacotherapy, vol. 42, pp. 447-450, and in Advances in CancerResearch, vol. 50, pp. 26-30 (1987).

The phrases “chemical substance selected from the group consisting oftumor promoters and tumor initiatory” is intended to encompass thosecompounds specifically named in Exhibits B-D. Those compounds which fallunder the definitions provided in EMF Health Report, vol. 1, No. 2(1993), and

which a worker of ordinary skill in the art would have known to be afunctional equivalent of a phorbol ester tumor promoter or adimethylbenz[α]anthracene tumor promoter, are also included.

SUMMARY OF THE INVENTION

A first object of this invention Is to evaluate a series of FDA-approvedsynthetic food colorants for inhibitory effects on carcinogenesis. Thecolorants were tested using in vitro assays for inhibition ofEpstein-Barr virus early antigen (EBV-EA) induction in cells exposed tothe tumor promotor 12-O-tetmdecanoylphorbol-13-actate (TPA). Highinhibitory activity toward EBV-EA induction is known to be indicativethat a composition has potential utility as an anti-tumor promoting orcancer chemopreventive agent. Epstein-Barr virus genome-carryinglumphobastoid cells (Raji cells) were used in the in vitro studies.

Synthetic food colorants which were found to be particularly effectiveas inhibitors of EBV-EA induction include:

a) sulfonated azo compounds having formula VIII:

wherein A is

and wherein R₁is —H lower alkyl lower alkoxy, —CO₂H, or —SO₃M; ; R₂ is—H, lower alkyl, or —SO₃M; R₃ is —H, lower alkyl, or —SO₃M; R₄ is —H,—CO₂M, or —SO₃M, R₅ and R₆, which may be the same or different areindependently selected from the group consisting of —H, —OH, —NH₂,—CO₂M, or —SO₃M; L is —H, lower alkyl, or lower hydroxyalkyl, and M is—H an alkali metal ion, or an alkaline earth metal cation;

b) colorants of formula IX and alum lake colorants derived fromcolorants of formula IX.

wherein X is —NH— or —S—, R₇ is —H or lower alkyl; R₈ is —H or —SO₃M; R₉is —H or —Cl; and M is —H or an alkali metal cation;

c) colorants of formula X:

wherein R₁₀ is —H or —OH and each R₁₁ is a lower alkyl group;

d) fluorescein derivatives of formula XI:

wherein Y is —H or —Br, and

fluorescein derivatives of formula XII:

wherein Z is —H, —Br or —I;

e) anthroquinone derivatives including dyes of fomula XIII:

wherein R₁₂ is —H or lower and and L is —OH or

carmine (XIV); and carminic acid (XV);

and

a pyrene derivative of formula XVI:

Although other synthetic colorants were effective as well, aromatic azocompounds, or axo dyes, which contain acidic or anionic groups appear tobe of particular utility in inhibation of carcinogenesis. Some of thesynthetic colorants which exhibited measurable inhibition of EBV-EAinduction were then bioassayed to determine whether they inhibitedTPA-promoted carcinogenesis in epidermal tissue. It was found that thecolorants which were tested in the bioassay appear to measurably inhibitformation of a chemically-promoted skin tumors.

A second object of the invention is to evaluate a series of plantextracts for inhibitory effects on carcinogenesis. The in vitroinhibitory effects of beet root extract and of grape extract onEpstein-Barr virus early antigen induction in Raji cells exposed to theminor promotor TPA were assayed, and found to show significantanti-tumor promoting activity. In fact, the inhibitory activity of beetroot extract was found to be greater than that shown by extracts of redbell peppers, red onion skin, paprika, and cranberries. In vivo studiesof carcinogenesis inhibition by beet root extract were then performed.When beet root extract was given orally to mice. formation of skintumors promoted by topical application of TPA or by exposure to UVradiation was significantly reduced. Also, beet root extract givenorally reduced the incidence of glycerol-promoted pulmonary tumorformation.

Research was also done on natural food colorants extracted from tumeric,annatto seeds, and paprika. These colorants are commercially availablein a variety of formulations in aqueous, vegetable oil, or propyleneglycol vehicles. These vehicles may also contain emulsifiers and/ordispersants such as polysorbate 80 and lecithin. Recent studies havereported that colorants derived from tumeric, annatto, and paprikainhibit carcinogenesis. In the work described herein, the anti-tumorpromoting activity of over thirty natural colorant formulation wasinvestigated. Many Of these Formulations were found to exhibitsignificant in vitro inhibition of EBV-EA activation in Raji cells.However, many of these formulations exhibited cytotoxicity, suggestingthat the use of these compositions to inhibit tumor formation may posehazards to human health. Fortunately, some compositions which inhibitedEBV-EA activation without exhibiting cytotoxicity were identified. Themost effective of these compositions were;

a) a mixture of natural extractives of annatto seeds and tumeric withpolysorbate 80, potassium hydroxide, and propylene glycol; and

b) a natural extractive of paprika in vegetable oil.

In vivo studies indicate that oral feeding of paprika and annattoextracts acts to inhibit TPA-induced skin tumor formation. They werealso found to be effective anti-inflammatory agents.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the method used to perform a bioassay for anti-tumorpromoting activity using a two-stage mouse skin carcinogenesis test.

FIG. 2 shows the inhibitory effect of the synthetic colorants tartrazineand erythrosine B on TPA-promoted mouse skin tumor promotion.

FIG. 3 shows the inhibitory effect of beetroot extract (betanin) onTPA-promoted mouse skin tumor promotion.

FIG. 4 shows the inhibitory effect of beetroot extract (betanin) onultraviolet B radiation-promoted mouse skin tumor promotion.

FIG. 5 shows the spleens of mice subjected to UV radiation treatment fortwenty weeks. Those from mice maintained on water alone show pronouncedenlargement (splenomegalia); those from mice maintained on watercontaining 0.0025% betanin do not.

FIG. 6 shows the inhibitory effect of natural extracts of annatto seedsand paprika on TPA-promoted mouse skin tumor promotion.

DETAILED DESCRIPTION OF THE INVENTION 1. In Vitro Studies on Inhibitionof EBV-EA Activation

1.1 Materials and Methods

In vitro studies were carried out using an Epstein-Barr virus (EBV)early antigen induction assay performed in the following manner. EBVgenome-carrying lymphoblastoid cells (Raji cells) were cultivated in anRMPI 1640 medium. The indicator cells were incubated at 37° C. for 48hours in 1 ml of the medium. The medium also contained the tumorpromoter TOA (20 ng/mi, or 32 pM), the tumor co-inducer butyric acid (4mM), and a, known amount of test dye in dimethylsulfoxide (DMSO). Smearswere then made from the cultured cell suspension. The cells exhibitingEBV activation in each smear were stained by high titer EBV-positivesera from nasopharyngeal carcinoma patients and detected usingconventional indirect immunofluorescence techniques. In each assay, atleast 500 cells were counted and the experiments were repeated at leasttwice. The average EA induction of each assay was compared to that of apositive control experiment. In the control experiment, Raji cells wereincubated at 37° C. for 48 hours in 1 ml of the medium. The medium alsocontained the tumor promoter TPA (20 ng/ml, or 32 pM) and the tumorco-inducer butyric acid (4 mM). In the control experiments, EA inductionwas normally around 30%.

In order to determine whether or not the synthetic colorants tested inthe EBV-EA activation tests exhibited unacceptable cytotoxicity, cellviability tests were performed using the trypan-blue staining me hod.After the EBV-EA activation test, 0.1 ml of a suspension of treatedcells from the EA induction test in phosphate buffer solution wasstained with 0.1 ml of a 0.25% trypan-blue solution. Dying cells weredyed blue. Non-dyed cells were counted. If more than 40% of cells weredyed blue (that is, fewer than 60% of the cells were living, viablecells), the cytotoxicity of the test compound used in the EA inductiontest was judged to be unacceptably high.

The synthetic colorants which were tested for inhibition of EBV-EAinduction using the assay techniques described above were obtained asgifts from the following sources:

a) Dr. A. Weiss

Food and Drug Administration

Center for Food Safety and Applied Nutrition Office of Cosmetics andColors

Washington, D.C.

b) Cromoton and Knowles Corp.

Mahway, N.J.

c) Hilton Davis Co.

Cincinnati, Ohio

d) Pylam Products Co., Inc.

Garden City, N.Y.

The plant-derived colorant formulations which were tested for inhibitionof EBV-EA activation by the described techniques are listed in Tables 1and 2. Table 1 lists colorant compositions derived from vegetableextracts, including beet roots, bell peppers, and onion skin. Table 2lists a series of colorant formulations derived from turmeric, annattoseeds, and/or paprika, together with some purified pigments which weretested for comparison. Curcumin and the colorant formulations derivedfrom turmeric, annatto seeds, and/or paprika were made available to usas gifts from Kalsec, Inc., Kalamazoo, Mich. Bixin, capsanthin, andbeetroot extract (betanin) were purchased form Tokyo Kasei Co., Japanthrough TCI America, Portland, Ore. USA. Grape extract was supplied byPenta Manufacturing, a division of Penta Internations Corporation,Fairfield, N.J. Beetroot extract (betanin) , grape extract, andcapsanthin (derived from paprika) were used without further processing.Red onions, cranberries, long red bell peppers (12 cm long, 7 cm wide atthe top, and 5 cm wide at the bottom) , short red bell peppers (10 cmlong, 7 cm wide at the top, and 4.5 cm wide at the bottom), purple bellpeppers, green bell peppers, and light yellow green bell peppers werepurchased at a supermarket in Potomac, Md. and used to prepare extracts.

To prepare extracts, the dry skin of onion and the other plant materials(cranberries, long red bell peppers, short red bell peppers, purple bellpeppers, green bell peppers, and light yellow green bell peppers) wereseparately crushed in a blender. In each case, a weighed amount ofmaterial was mechanically shaken in a 250 ml flask for 1 hr with 95%ethanol. The mixture was thereafter centrifuged and the supernatant wasdecanted and filtered. The insoluble residual material was twicereextracted for 20 min by stirring with half the volume of the samesolvent as used during the first extraction. The combined extracts wereevaporated under a vacuum and weighed, and the percentage yields wererecorded. The yield of the dry red onion skin alcoholic extract was13.2%; that from cranberry was 7.06%; from short red bell pepper 7.06%;and from long red bell pepper 10%.

1.2 Results and Discussion

A wide variety of synthetic colorants were tested for in vitroinhibition of Epstein-Barr virus early antigen induction by TPA. Thetested synthetic colorants include:

a) the following aromatic azo compounds:

b) indigo and the following related compounds:

c) the following substituted triphenylmehtyl compounds:

d) the following substituted fluorescein compounds:

e) the following anthroquinone derivatives:

f) the pyrene derivative D&C Green No. 8 (XVI); and

g) the following quinine derivatives:

Each synthetic colorant was tested for activity as an inhibitor ofEBV-EA induction in Raji cells according to the previously describedmethod, and the results are recorded in Tables 3 and 4. In each of thesetables, the entries have been arranged roughly in order of inhibitoryactivity toward EBV-EA induction, with the most active colorants beingentered first. The tested synthetic colorants have been classified as:

strongly effectively EBV-EA induction inhibitors if all EBV-EA inductionis inhibited at a mole ratio of colorant to tumor promotor of 1000 orless;

moderately effective EBV-EA induction inhibitors if the number of Rajicells exhibiting signs of EBV-EA induction in the presence of thecolorant (mole ratio of colorant to tumor promotor=1000) is greater than0% and less than 40% of the number of Raji cells exhibiting signs ofEBV-EA induction in the absence of the colorant; and

weakly effective or ineffective EBV-EA induction inhibitors if thenumber of Raji cells exhibiting signs of EBV-EA induction in thepresence of the colorant (mole ratio of colorant to tumor promotor=1000)is greater than 40% of the number of Raji cells exhibiting signs ofEBV-EA induction in the absence of the colorant.

The phenylazonaphthalene derivative D&C Red #33 was found to be the mostactive among the colorant samples tested in vitro which are included inTable 3, followed by the azo compounds FD&C Yellow #5 and D&C Red #7. Infact, it appears that five of the ten most effective colorants includedin Table 3 have azo groups. The results in Table 4, like those in Table3, indicate that an azo compound is the most active of the colorantsincluded. All of the azo compounds, with the exception ofphenazopyridine hydrochloride, appear to show at least moderateanti-tumor promoting activity. While it is interesting to note thatphenazopyridine hydrochloride is the only tested azo compound which hasno acidic or anionic groups, it is unclear whether this is the cause ofits low anti-tumor promoting activity, or merely an interestingcoincidence. This would seem to indicate that azo compounds may havetherapeutic utility as anti-tumor promoting agents.

The fluorescein colorants showed no clear pattern in their activity,with D&C Red #22 (the disodium salt of tetrabromofluorescein) showingstrong anti-tumor promoting activity, while D&C Yellow #8, D&C Orange #5(dibromofluorescein), D&C Yellow #7 (fluorescein), and D&C Orange #10(diiodofluorescein) exhibit moderate anti-tumor promoting activity.Tetrachlorofluorescein, D&C Red #19 (Rhodamine B), D&C Red #27(tetrachlorotetrabromofluorescein), D&C Red #28 (the disodium salt ofD&C Red #27), and FD&C Red #3 (the disodium salt oftetraiodofluorescein) were among the least active of the compoundsstudied.

As for the remaining compounds, the anthroquinone derivatives (carmine,carminic acid, D&C Green No. 5, and Ext. Violet No. 2) showed stronginhibitory activity toward EBV-EA induction. The quinoline derivativeD&C Yellow #11 was an extremely inactive inhibitor of EBV-EA induction.Surprisingly, however, D&C Yellow #10 (a sulfonated derivative of D&CYellow #10), while not a strong EBV-EA inhibitor, did show significantanti-tumor promoting activity. The pyrene derivative (D&C Green No. 8)and the triphenylmethyl derivatives (FD&C Blue No. 1 and FD&C Green No.3) were moderately active inhibitors of EBV-EA induction. Theindigo-type compound FD&C Blue No. 2 was a strongly effective anti-tumorpromoting agent; however, other indigo-type compounds, D&C Blue No. 6and alum lake of D&C Red No. 30, were only moderately effective at best.The synthetic colorants studied all exhibited low cytotoxicity.

The vegetable extracts listed in Table 1 were also tested for in vitroinhibition of Epstein-Barr virus early antigen induction by TPA, and theresults are recorded in Table 5. It is found that the beetroot extract,containing high levels of betanin, inhibits EBV-EA induction by TPAquite strongly. In fact, beetroot extract is more effective at EBV-EAinhibition than capsanthin, a colorant found in paprika. Grape extractwas also found to be an effective inhibitor of EBV-EA induction,exhibiting an activity which appears to be greater than that ofcapsanthin, although not as great as that of beet root extract. Redonion skin extract, although possessing some inhibitory activity towardTPA-induced EBV-EA induction, was found to be less active than beetrootextract, grape extract, or capsanthin, Cranberry and long red bellpepper inhibited EBV-EA induction less strongly than red onion skinextract. Interestingly, long red bell pepper was the least active of thesix extracts studied, while short red and purple bell peppers were themost active, exhibiting slightly greater activity than beetroot extract.The activity of the green and light yellow green bell peppers wereroughly as active as red onion skin. It is unclear why the differenttypes of bell pepper extracts have such widely varying activity. None ofthe vegetable extracts studied showed unacceptable levels ofcytotoxicity.

From the above data, it would appear that the beetroot extract,containing betanins as the primary colorants, is an effective inhibitorof EBV-EA induction. More particularly, it seems that betanins are moreeffective inhibitors of EBV-EA induction than capsanthin, one of theprimary carotenoid colorants in paprika, or the anthocyanins, theprimary colorants in cranberry and red onion skin. The data presenteddoes not allow us to make a clear comparison between the inhibitoryactivity of betanins and the carotenoids, the primary colorants in bothlong and short red bell peppers. Nevertheless, the results do seem toindicate that beetroot extracts containing betanins possess potential asinhibitors of TPA-induced tumor formation.

With regard to the vegetable extracts discussed above, it must be notedthat it is unproven that the anti-tumor promoting activity is due tocolorant compounds. Since the extracts were crude, the anti-tumorpromoting activity may be due to a non-colorant compound present in anextract.

The thirty-seven colorant formulations derived from paprika, turmeric,and/or annatto seeds listed in Table 2 were also tested for in vitroinhibition of Epstein-Barr virus early antigen induction by TPA. Theresults of these tests are recorded in Table 6. Based on the data inTable 6, the colorant formulations have been classified as:

a) strongly effectively EBV-EA induction inhibitors if:

i) all EBV-EA induction is inhibited at a colorant concentration of 100micrograms/ml or less; and

ii) the number of Raji cells exhibiting signs of EBV-EA induction in thepresence of the colorant formulation (colorant concentration =10micrograms/ml) is less than 60% of the number of Raji cells exhibitingsigns of EBV-EA induction in the absence of the colorant;

b) moderately effective EBV-EA induction inhibitors if:

i) the number of Raji cells exhibiting signs of EBV-EA induction in thepresence of the colorant formulation (colorant concentration=100micrograms/ml) is greater than 0% and less than 50% of the number ofRaji cells exhibiting signs of EBV-EA induction in the absence of thecolorant; or

ii) all EBV-EA induction is inhibited at a colorant concentration of 100micrograms/ml; and the number of Raji cells exhibiting signs of EBV-EAinduction in the presence of the colorant formulation at a colorantconcentration of 10 micrograms/ml is greater than 60% of the number ofRaji cells exhibiting signs of EBV-EA induction in the absence of thecolorant; and

c) weakly effective or ineffective EBV-EA induction inhibitors if thenumber of Raji cells exhibiting signs of EBV-EA induction in thepresence of the colorant formulation at a colorant concentration of 100micrograms/ml is greater than 50% of the number of Raji cells exhibitingsings of EBV-EA induction in the absence of the colorant. By applyingthis system to the results presented in Table 6, the colorantformulations listed in Table 2 have been classified as follows:

strongly active: 1, 2, 4, 9, 11, 15, 16, 19, 20, 21, 22, 24, and 35

moderately active: 3, 5, 6, 7, 12, 13, 14, 17, 18, 23, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 36, and 37

weakly active or inactive: 8 and 10

From the data in table 6, it appears that colorant formulation No. 1, aturmeric extract containing 85-97% curcumin which inhibits all EBV-EAinduction at a colorant concentration of 10 micrograms/ml, is the mosteffective of these compositions as an inhibitor of EBV-EA induction.However, when cell toxicity studies were conducted, it was found that athigh concentration of the colorant (100 micrograms/ml), only 20% of theRaji cells used in the EBV-EA activation assay survived exposure tocolorant formulation No. 1. In fact, over 45% of the tested compositionsderived from paprika, turmeric, and/or annatto seeds showed unacceptablyhigh levels of cytotoxicity (see Table 6). Even if these cytotoxiccompounds possess sufficient inhibitory activity toward EBV-EA inductionto suggest that they have utility as anti-tumor promoting or cancerchemopreventive agents, the high levels of cytotoxicity raise humanhealth concerns that would argue against their therapeutic use.

Fortunately, several of the compositions derived from paprika, turmeric,and/or annatto seeds showed acceptably low levels of cytotoxicity towardRaji cells in the EBV-EA activation test. At least 60% of the Raji cellsexposed to these colorant formulations at a colorant concentration of100 micrograms/ml survived. Based on the data in Table 6, thenon-cytotoxic colorant formulations have been classified in thefollowing manner with regard to their inhibitory activity towardTPA-induced EBV-EA induction:

strongly active: 4, 9, 15, 16, 21, and 35

moderately active: 7, 13, 14, 17, 25, 26, 31, 32, 33, and 34

weakly active or inactive: 8 and 10

Out of the six most effective non-cytotoxic compositions showinganti-tumor activity, two (No. 4 and No. 35) are composed of naturalextractives of annatto seeds and turmeric in a vehicle made frompolysorbate 80, KOH, and propylene glycol. The activity of thisformulation against EBV-EA induction was greater than that of theannatto seed-derived pigment bixin itself (colorant formulation No. 36)but somewhat less than that of turmeric containing 85-97% curcumin(colorant formulation No. 1). Formulations No. 4 and No. 35 also showmuch lower cytotoxicity than formulations No. 1 and No. 36, suggestingthat therapeutic use of a blend of natural extractives of annatto seedsand turmeric as anti-tumor promoting or cancer chemopreventive agentsmay pose lower health risks than use of purified pigments from eitherturmeric or annatto seeds alone. Similarly, colorant formulation No. 21,composed of natural extractives of paprika with vegetable oil showedhigher inhibitory activity toward EBV-EA induction than that of the purepaprika-derived pigment capsanthin in vegetable oil (colorantformulation No. 37), coupled with low cytotoxicity. Colorant formulationNo. 15, composed of natural extractives of annatto seeds with propyleneglycol, water, and KOH, appears to show greater anti-tumor promotingactivity and slightly lower cytotoxicity than colorant formulation No.36, containing the pigment bixin. Interestingly, the activity againstEBV-EA induction shown by some natural extractives from paprika andannatto seeds appears to be greater than that of the purified pigmentsfrom paprika and annatto seeds.

Other colorant formulations that appear from this data to beparticularly effective inhibitors of EBV-EA induction include:

natural extractives of annatto seeds and turmeric in a medium comprisingvegetable oil and fumed silicon dioxide; and

extractives of annatto seeds having a norbixin content of 2.6-4.0 wt. %in a medium comprising water and KOH.

2. In Vivo Studies on Inhibition of Carcinogenesis in Mice

2.1 Materials and Methods

Two-stage mouse skin carcinogenesis tests were conducted on mice. Eachgroup of mice was composed of 15 mice housed 5 per cage. Basal diet andtap water were available ad libitum throughout the experiment followingthe method of Tokuda eta l. (Tokuda et al., Oncology, 48, 77-80 [1991]).

Studies on inhibition of TPA-induced skin carcinogenesis by syntheticcolorants were conducted on 6-week-old female mice, specificpathogen-free ICR strain, using the following procedure. The back ofeach mouse was shaved with surgical clippers. Each mouse was thentreated topically with 390 nmol 7,12-dimethylbenz[a]anthracene (DMBA) in0.1 ml acetone. One week before TPA-treatment, each mouse in a testgroup was treated with an acetone solution of the test dye (85 nM). Acontrol group was not treated with dye. The incidence of papillomas wasobserved weekly for twenty weeks.

Studies on inhibition of TPA-induced skin carcinogenesis by plantextracts and plant pigments were conducted on 6-week-old female mice,specific pathogen-free ICR strain, using the following procedure. Theback of each mouse was shaved with surgical clippers. Each mouse wasthen treated topically with 390 nmol 7,12-dimethylbenz[a]anthracene(DMBA) in 0.1 ml acetone. After 1 week, each mouse received topically1.7 nmol TPA in 0.1 ml acetone twice a week for twenty weeks. During TPAtreatment, a test group of mice was given drinking water containing adefined test compound, while a control group was given water containingno test compound. For betanin studies a 0.0025% solution of beetrootextract was given orally to the mice (2.5 mg/100 ml H₂O). In studies ofpaprika and annatto seed extract formulations, the formulations weredissolved in a small volume of ethanol and diluted by water (2.5 mgextract/100 ml H₂O to provide a 0.0025% solution). The incidence ofpapillomas was observed weekly for twenty weeks.

A similar test was performed for inhibition of ultravioletradiation-induced skin carcinogenesis. The mice used were 6-week-oldfemale hairless mice, specific pathogen-free Hos:HR-1 strain. Each mousewas treated topically with 390 nmol 7,12-dimethylbenz[a]anthracene(DMBA) in 0.1 ml acetone. After 1 week, each mouse was exposed to UVBradiation for eight minutes twice a week for twenty weeks. Theultraviolet radiation was applied at a dosage of 3,430 J/m². During TPAtreatment, a test group of mice was given drinking water containing0.0025% beetroot extract (2.5 mg extract/100 ml of water), while acontrol group was given water containing no beetroot extract. Theincidence of papillomas was observed weekly for twenty weeks. The lampsused for irradiation of the test mice were Toshiba FL20 S.E. lamps,emitting UV radiation in the 280-320 nm range, with a peak at 305 nm.

Bioassays for inhibition of glycerol-promoted pulmonary tumor formationin mice were also performed. The animals used were ICR male mice. Ineach group of 15 mice, 0.3 mg of 4-nitroquinoline 1-oxide (4NQO) wasinjected into the back of each mouse as an initiator. After 5 weeks, afirst group of mice was maintained with an 8% glycerol solution as itsonly source of drinking water. A second group was maintained with0.0025% betanin solution (2.5 mg/100 ml of 8% glycerol). A third,control group was maintained with water alone. Two additional groupswhich ere not subjected to treatment with 4NQO were also tested. One ofthese non-4NQO-treated groups was maintained with water alone; the otherwas maintained with 8% glycerol. After 25 weeks, all mice weresacrificed by cervical dislocation and autopsy was performed. Lungtumors (adenoma) were counted after separation of each pulmonary lobe.Statistical analysis was done using Student's t-test.

The standard mouse ear edema method was followed for the evaluation ofanti-inflammatory activity. Indomethacin was used as a control.Seven-week-old ICR male mice were used for the test. On the left side ofa mouse's ear, 10 micrograms TPA in 10 microliters acetone were applied.On the right side of the mouse's ear, 10 micrograms TPA and a definedamount of a test compound in 10 microliters acetone were applied. Aftera defined period of time, a portion of TPA-treated mouse ear was removedand weighed. At the same time, a portion of TPA and testcompound-treated mouse ear of the same size and shape as the portion ofTPA-treated mouse ear was removed and weighed. Any increase in weightover a section of non-treated mouse ear was taken as a measure ofinflammation.

Six-week-old ICR mice and 7-week-old ICR mice were purchased from JapanSLC, Inc., Shizuoka, Japan. Six-week-old Hos:HR-1 mice were purchasedfrom Hoshino Animal Factory, Saitama, Japan. Acetone, DMBA, glycerol,4NQO, and TPA were purchased from Wako Pure Chemical Industries, Osaka,Japan.

2.2 Results and Discussion

Two of the synthetic colorants tested for in vitro inhibition ofTPA-induced EBV-EA induction were also studied in vivo to determinewhether they inhibited TPA-induced tumor formation in mice. The studieswere conducted using a two-stage mouse skin carcinogenesis bioassay. Thecolorants studied were tartrazine (FD&C Yellow #5), which was among themost active colorants tested in the in vitro EBV-EA induction assay, anderythrosine B (FD&C Red #3), which was one of the least activecolorants.

The results are presented in FIG. 2. FIG. 2 shows the percentage ofpapillomas (or, more precisely, the percentage of mice havingpapillomas) formed with and without colorant treatment over a period oftwenty weeks. The control animals, without the colorant treatment, show100% incidence of papillomas in less than ten weeks. The test animals,which have been treated with colorant, take 15 weeks to show even 80%papilloma formation. This is also seen in the number of papillomasformed per mouse over a fifteen week period (FIG. 2). AS seen, both thecolorants appear to cause a 40% reduction in the number of papillomasper mouse. Thus, tartrazine and erythrosine B both show inhibitoryactivity toward TPA-induced two stage skin carcinogenesis.

Beetroot extract (betanin) was also tested for inhibitory activitytoward TPA-induced tumor formation in mice using a two-stage mouse skincarcinogenesis bioassay. As shown in FIG. 3, oral ingestion of betaninin ICR mice inhibited TPA-induced promotion of mice skin tumors. Boththe percentage of mice having papillomas and the number of papillomasformed per mouse are significantly lower in mice given a betaninsolution to drink than they are in mice given substantially pure water.

Oral ingestion of betanin in Hos:HR-1 mice also acts to inhibit UVBradiation-promoted skin carcinogenesis, as shown in FIG. 4. The controlanimals, drinking water alone, show 100% incidence of papillomas in lessthan ten weeks of radiation treatment. In the same period, only 25% ofthe test animals, drinking water containing 0.0025% betanin, show anyincidence of tumors. The test animals take 15 weeks to show even 80%papilloma formation. Evidence of anti-tumor activity is also seen in thenumber of papillomas formed per mouse over a twenty week period. Oralfeeding of betanin in drinking water causes a 40% reduction in thenumber of tumors per mouse, as compared to the number of tumors permouse in a control group of mice maintained on water alone. The mice inthe control group were also found to show splenomegalia (“hypertrophiaspleen”). The test animals, which were fed betanin in drinking water,did not show splenomegalia. FIG. 5 provides a visual comparison of thespleens of mice subjected to UVB radiation treatment for twenty weekswhile being maintained on water alone (positive control) and the spleensof mice subjected to radiation treatment while being maintained on watercontaining 0.0025% betanin. This would seem to indicate that betanin isan effective inhibitor of ultraviolet B irradiation-induced skin tumorformation, and also affords protection against UVB irradiation-inducedsplenomegalia.

Beetroot extract was also tested for inhibitory activity towardpulmonary tumor formation. As shown in Table 7, oral ingestion ofbeetroot extract in drinking water containing 8% glycerol leads to a 60%reduction of lung tumors in 4NQO-treated mice, compared to 4NQO-treatedmice maintained an an 8% solution of glycerol alone. These findings,together with the findings on skin tumor formation, indicate thatbeetroot is a useful cancer preventive vegetable.

Two of the colorant formulations derived from turmeric, annatto seeds,and/or paprika were also tested for inhibition of TPA-induced tumorformation in mice, and the results are shown in FIG. 6. A control groupof mice maintained on water alone showed 100% incidence of skin tumorsin ten weeks. By comparison, a test group of mice maintained on watercontaining 0.0025% colorant formulation #21, composed of naturalextractives of paprika with vegetable oil, showed only 80% incidence oftumors after 15 weeks. A 60% reduction in the number of tumors per mousewas also observed at the end of a twenty week period. Another test groupof mice was maintained on water containing 0.025% colorant formulation#22, composed of natural extractives of annatto seeds with vegetableoil. Formulation #22 was less effective than formulation #21, showing80% incidence of tumors after only 13 weeks. More tumors per mouse wereseen after twenty weeks in mice fed formulation #22 than in those fedformulation #21. Additionally, the in vitro results presented in Table 6indicate that formulation #21 has significantly lower cytotoxicity thanformulation #22. This data would seem to indicate that a composition ofnatural extractives of paprika with vegetable oil may have potentialtherapeutic utility as an anti-tumor promoting or cancer chemopreventiveagent.

Formulation #21 and formulation #22 were also tested foranti-inflammatory activity by the mouse ear edema method. Bothformulations exhibited anti-inflammatory activity, with formulation #21showing greater activity than #22. The results are provided in Table 8.As can be seen, the extractive of paprika in vegetable oil (formulation#21) exhibited comparatively strong anti-inflammatory activity ascompared to indomethacin. The extractive of annatto seeds with vegetableoil (formulation #22) showed lower activity than #21. It is possiblethat paprika extractives may be therapeutically useful anti-inflammatoryagents.

TABLE 1 Vegetable-Derived Colorant Extracts Tested for EBV-EA InhibitionExtract Colorant Ingredient Beet Root Betanins Red Onion SkinAnthocyanins Paprika Capsanthin Cranberry Anthocyanins Short Red BellPeppers Carotenoids Long Red Bell Peppers Carotenoids Grape Anthocyanins

TABLE 2 Turmeric-, Paprika-, and/or Annatto Seed-Derived ColorantFormulations Tested for EBV-EA Inhibition Colorant CompositionFormulation No. (As provided in the product literature) 1 Turmeric,Curcumin Content 85-97% 2 Turmeric, Curcumin Content 29-31% 3 NaturalExtractives of Turmeric with not more than 80% Polysorbate 80, KOH, andAntifoam 4 Natural Extractives of Annatto Seeds and Turmeric withPolysorbate 80, KOH, and Propylene Glycol 5 Natural Extractives ofAnnatto Seeds and Turmeric with Vegetable Oil and Fumed Silicon Dioxide6 Natural Extractives of Annatto Seeds and Turmeric with Vegetable Oil,Mono-, Di-, and Triglycerides, 7.3% BHT, and Fumed Silicon Dioxide 7Natural Extractives of Turmeric and Paprika with Vegetable Oil and FumedSilicon Dioxide 8 Natural Extractives of Annatto Seeds with Water andKOH with Norbixin Content of 1.04-1.16% 9 Extractives of Annatto Seedswith Water and KOH with Norbixin Content of 2.6-2.8% 10 Extractives ofAnnatto Seeds with Propylene Glycol and KOH with Bixin Content of2.48-2.67% 11 Natural Extractives of Turmeric with Lecithin, Mono- andDiglycerides, and Vegetable Oil 12 Natural Extractives of Annatto Seedsand Turmeric with Polysorbate 80, KOH, and Propylene Glycol 13 NaturalExtractives of Annatto Seeds with Vegetable Oil 14 Natural Extractivesof Annatto Seeds with Vegetable Oil, Mono-, Di-, and Triglycerides, andKOH 15 Natural Extractives of Annatto Seeds with Propylene Glycol,Water, and KOH 16 Natural Extractives of Annatto Seeds and Turmeric withVegetable Oil and Fumed Silicon Dioxide 17 Natural Extractives ofAnnatto Seeds with Propylene Glycol, Polysorbate 80, and KOH with BixinContent of 2.48-2.67% 18 Natural Extractives of Turmeric with Mono-,Di-, and Triglycerides and Propylene Glycol 19 Natural Extractives ofPaprika and Turmeric with Vegetable Oil and Fumed Silicon Dioxide 20Natural Extractives of Turmeric with Propylene Glycol 21 NaturalExtractives of Paprika with Vegetable Oil 22 Natural Extractives ofAnnatto Seeds with Vegetable Oil 23 Natural Extractives of Paprika withMono- and Di-glycerides, Lecithin, and Vegetable Oil 24 NaturalExtractives of Turmeric with Polysorbate 80 25 Natural Extractives ofAnnatto Seeds and Turmeric with Vegetable Oil 26 Natural Extractives ofAnnatto Seeds and Paprika with Vegetable Oil and Fumed Silicon Dioxide27 Natural Extractives of Turmeric with Vegetable Oil 28 NaturalExtractives of Annatto Seeds with Vegetable Oil and Fumed SiliconDioxide 29 Natural Extractives of Annatto Seeds with Water and KOH 30Natural Extractives of Annatto Seeds with Vegetable Oil, Mono- andDi-glycerides and Lecithin 31 Natural Extractives of Annatto Seeds withPropylene Glycol, Water and KOH 32 Natural Extractives of Annatto Seedswith Water and KOH with Norbixin Content of 3.65-4.0% 33 NaturalExtractives of Annatto Seeds with Vegetable Oil, Mono-, Di-, andTriglycerides, and KOH 34 Natural Extractives of Annatto Seeds andPaprika with Vegetable Oil, Mono-, Di-, and Triglycerides, and KOH 35Natural Extractives of Annatto Seeds and Turmeric with Polysorbate 80,KOH, and Propylene Glycol 36 Bixin/Annatto Extract 37 Capsinthin/PaprikaExtract in Vegetable Oil

TABLE 3 Inhibitory Effect of Synthetic Colorants on TPA-Induced EBV-EAInduction % relative to control (% viability)* Colorant Concentration(mol ratio/TPA) Colorant 1000 500 100 10 D&C Red # 33  0 (70) 15.5 42.188.1 FD&C Yellow # 5  0 (70) 37.1 65.8 89.0 D&C Red # 7  0 (70) 44.765.0 89.5 D&C Green # 5  0 (70) 0 66.5 93.7 Olsal  0 (60) 11.2 65.9 100FD&C Yellow # 6  0 (70) 64.8 72.0 100 Sulfasal  0 (60) 26.9 72.0 100Carminic Acid  0 (70) 65.8 88.0 100 D&C Orange # 4  0 (70) 18.6 68.3 100D&C Red # 22  0 (60) 32.5 73.9 100 Ext. D&C  0 (70) 0 87.0 100 Violet #2 Carmine  0 (70) 26.4 91.5 100 FD&C Blue # 2  0 (70) 43.7 91.4 100 FD&CRed # 40 19.6 (70) 34.8 75.5 100 D&C Orange # 5 24.7 (70) 57.2 82.6 100D&C Yellow # 7 33.8 (60) 54.0 76.3 100 D&C Green # 8 15.8 (60) 39.3 84.0100 FD&C Green # 3 17.9 (70) 66.2 82.9 100 D&C Yellow # 8 16.7 (60) 52.891.6 100 FD&C Blue # 1 12.8 (70) 66.0 93.5 100 D&C Yellow # 10 16.3 (60)57.0 100 100 D&C Red # 6 32.3 (70) 55.0 81.4 100 D&C Orange # 10 17.9(70) 81.3 100 100 D&C Blue # 6 25.1 (70) 84.9 100 100 Tetrachloro- 41.3(60) 63.9 82.0 100 fluorescein D&C Red # 19 43.8 (60) 69.2 85.9 100 D&CYellow # 8 46.2 (60) 67.3 86.9 100 Phenazo 47.2 (60) 74.6 93.1 100 D&CRed # 21 45.1 (70) 72.6 92.4 100 D&C Red # 28 53.8 (60) 77.4 100 100FD&C Red # 3 63.5 (70) 85.7 100 100 D&C Yellow # 11 81.5 (70) 100 100100 D&C Red # 27 88.8 (70) 100 100 100 *Values represent percentages ofRaji cells showing EBV-EA induction (error ≦± 3%), measured relative tothe positive control value (TPA 32 pmol = 100%). Values in parenthesesare viability percentages of Raji cells.

TABLE 4 Inhibitory Effect of Synthetic Colorants on TPA-Induced EBV-EAInduction % relative to control (% viability)* Colorant Concentration(micrograms/ml) Colorant 10 1 0.1 0.01 D&C Red # 39   0 (60) 61.7 88.2100 FD&C Red # 3 32.4 (70) 64.9 92.0 100 FD&C Blue # 1 32.6 (70) 67.790.3 100 FD&C Red # 4 25.6 (60) 87.2 100 — D&C Red 30, 21.4 (70) 90.5100 100 A1 Lake *Values represent percentages of Raji cells showingEBV-EA induction, measured relative to the positive control value (TPA32 pmol = 100%). Values in parentheses are viability percentages of Rajicells.

TABLE 5 Inhibitory Effect of Vegetable Dye Extracts on TPA-InducedEBV-EA Induction % relative to control (% viability)* ExtractConcentration (micrograms/ml) Extract 500 100 10 1 Beet Root 0 (60) 8.340 89.4 Grape** — 0 (70) 57.2 85.7 Capsanthin 0 (70) 19.5 54.3 87.5Cranberry — 45.1 90 100 Red Onion — 0 (60) 65.6 87.2 Skin Long Red Bell— 54.0 (70) 100 100 Pepper Short Red Bell — 0 (70) 25.0 80.4 PepperPurple Bell — 0 (70) 18.5 85.7 Pepper Green Bell — 0 (70) 63.9 100Pepper Light Yellow — 0 (70) 79.2 100 Green Pepper *Values representpercentages of Raji cells showing EBV-EA induction, measured relative tothe positive control value (TPA 32 pmol = 100%). Values in parenthesesare viability percentages of Raji cells. **At 0.1 micrograms/ml extractconcentration, the percentage of Raji cells showing EBV-EA induction,measured relative to the positive control value is 100%.

TABLE 6 Inhibitory Effect of Colorants Derived from Paprika, Turmeric,and/or Annatto Seeds on TPA-Induced EBV-EA Induction % relative tocontrol (% viability)* Colorant Colorant Concentration (micrograms/ml)Formulation 100 10 1 Cytotoxic? 1 0 (20) 0 (60) 67.2 yes 2 0 (10) 46.1(70) 89.4 yes 3 16.4 (0) 42.7 (60) 69.5 yes 4 0 (79) 48.3 67.2 no 5 28.7(0) 100 (60) 100 yes 6 36.7 (20) 85.3 (70) 100 yes 7 0 (70) 80.1 100 no8 90.6 (70) 100 100 no 9 0 (70) 44.7 100 no 10 72.8 (70) 93.8 100 no 110 (0) 45.9 (60) 93.2 yes 12 0 (0) 64.7 (60) 95.5 yes 13 33.9 (60) 58.0100 no 14 17.4 (60) 100 100 no 15 0 (60) 36.8 90.2 no 16 0 (60) 57.492.5 no 17 30.7 (70) 84.0 100 no 18 10.6 (0) 46.9 (70) 86.1 yes 19 0 (0)45.4 (70) 73.0 yes 20 0 (0) 58.9 (60) 80.4 yes 21 0 (60) 27.3 77.9 no 220 (0) 35.5 (60) 73.0 yes 23 15.7 (0) 59.4 (60) 93.7 yes 24 0 (0) 46.3(60) 89.6 yes 25 18.6 (80) 100 100 no 26 25.9 (70) 90.6 100 no 27 0 (20)82.7 (70) 100 yes 28 28.4 (30) 67.6 (70) 100 yes 29 0 (50) 93.0 100 yes30 0 (50) 75.4 100 yes 31 0 (80) 63.8 100 no 32 0 (70) 64.6 100 no 3332.6 (80) 66.3 84.9 no 34 45.2 (80) 73.7 92.8 no 35 0 (80) 20.4 85.6 no36 0 (50) 66.9 (70) 100 yes 37 19.5 54.3 87.5 unknown *Values representpercentages of Raji cells showing EBV-EA induction, measured relative tothe positive control value (TPA 32 pmol = 100%). Values in parenthesesare viability percentages of Raji cells.

TABLE 7 Incidences of pulmonary tumor in mice treated with betanin 8%glycerol % of mice Group Body wt intake (ml/day Total no. of No. oftumor/ with treatment (g) per mouse) tumors mouse tumors I. Water alone47.6 ± 4.8 8.0 0 0 0 II. 8% glycerol alone 56.4 ± 5.4 8.8 0 0 0Initiation + promotion III. 4NQO + water 46.6 ± 4.6 7.9 3 0.2 ± 0.1 6.6IV. 4NQO + 8% glycerol 55.2 ± 4.9 8.6 50 3.2 ± 0.5 100 V. 4NQO + 8%glycerol + 55.0 ± 5.1 8.6 14 0.9 ± 0.3 40 0.0025% betanin ^(a)Groups of15 mice were effective numbers at the end of the experiment. ^(b)Nostatistically differences were observed between groups by mean intake ofdrinking. ^(c)The increase in the body weight of the treated mice wasnot affected by treatment with betanin.

TABLE 8 Anti-Inflammatory Activity of Paprika and Annatto Seed ExtractsConc.* (μg/10 μl acetone) Indomethacin Kal-21 Kal-22 100 μg +++ ++ ++ 10 ++ + −  1 + − − +++ = strongest anti-inflammat.activity ++ = strongactivity + = weak activity − = no activity *On left side of the mouse'sear, 10 μg TPA in 10 μl acetone were applied. On right side of themouse's ear, 10 μg TPA in 10 μl acetone were applied, together with adefined amount of test compound in 10 μl acetone.

What is claimed is:
 1. A method of reducing the incidence of pulmonarytumor formation in animals exposed to a chemical selected from the groupconsisting of a tumor-promoting chemical and a tumor-initiatingchemical, wherein a reduction in the incidence of pulmonary tumorformation is measured in terms of a reduction in the number of tumorsper animal or a reduction in the percentage of animals which exhibittumor formation, said method comprising the steps of: obtaining a groupof animals which have been exposed to a defined amount of a chemicalselected from the group consisting of a tumor-promoting chemical and atumor-initiating chemical; and providing the group of animals withdrinking water containing an amount of betanins which is effective toreduce the incidence of pulmonary tumor formation in the group ofanimals, said betanins having been extracted from beetroot; wherein thedefined amount of the chemical is sufficient to increase the incidenceof pulmonary tumor formation in a group of animals which have beenexposed to the chemical, and have been provided with drinking watercontaining no betanins.
 2. The method of claim 1, wherein thetumor-promoting chemical is contained in the animal's drinking water. 3.The method of claim 2, wherein the tumor-promoting chemical is glycerol.4. The method of claim 1, therein the drinking water is an aqueoussolution containing 0.0025 wt. % betanins.
 5. The method of claim 3,wherein the drinking water is an aqueous solution containing 0.0025 wt.% betanins and 8 wt. % glycerol.
 6. The method of claim 1, wherein thetumor-initiating chemical is injected into each animal prior toadministration of the drinking water containing betanins.
 7. The methodof claim 6, wherein the tumor-initiating chemical is 4-nitroquinoline1-oxide.
 8. The method of claim 6, therein the drinking water is anaqueous solution containing 0.0025 wt. % betanins.
 9. The method ofclaim 1, wherein a tumor-initiating chemical is injected into eachanimal prior to administration of the drinking water containingbetanins, and wherein a tumor-promoting chemical is contained in theanimal's drinking water.
 10. The method of claim 9, wherein thetumor-initiating chemical is 4-nitroquinoline 1-oxide, and thetumor-promoting chemical is glycerol.
 11. The method of claim 6, thereinthe drinking water is an aqueous solution containing 0.0025 wt. %betanins.